Composition and method for the repair and regeneration of cartilage and other tissues

ABSTRACT

The present invention relates to a new method for repairing human or animal tissues such as cartilage, meniscus, ligament, tendon, bone, skin, cornea, periodontal tissues, abscesses, resected tumors, and ulcers. The method comprises the step of introducing into the tissue a temperature-dependent polymer gel composition such that the composition adhere to the tissue and promote support for cell proliferation for repairing the tissue. Other than a polymer, the composition preferably comprises a blood component such as whole blood, processed blood, venous blood, arterial blood, blood from bone, blood from bone-marrow, bone marrow, umbilical cord blood, placenta blood, erythrocytes, leukocytes, monocytes, platelets, fibrinogen, thrombin and platelet rich plasma. The present invention also relates to a new composition to be used with the method of the present invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/896,912 filedJun. 29, 2001 now abandoned, which claims the benefit under 35 U.S.C. §119(e) of United States Provisional Application Nos. 60/214,717 filedJun. 29, 2000.

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The invention relates to a composition and method of application toimprove the repair and to regenerate cartilaginous tissues and othertissues including without limitation meniscus, ligament, tendon, bone,skin, cornea, periodontal tissues, abscesses, resected tumors, andulcers.

(b) Description of Prior Art

1) The Cartilage Repair Problem:

Cartilage: Structure, Function, Development, Pathology

Articular cartilage covers the ends of bones in diarthroidial joints inorder to distribute the forces of locomotion to underlying bonestructures while simultaneously providing nearly frictionlessarticulating interfaces. These properties are furnished by theextracellular matrix composed of collagen types II and other minorcollagen components and a high content of the proteoglycan aggrecan. Ingeneral, the fibrillar collagenous network resists tensile and shearforces while the highly charged aggrecan resists compression andinterstitial fluid flow. The low friction properties are the result of aspecial molecular composition of the articular surface and of thesynovial fluid as well as exudation of interstitial fluid during loadingonto the articular surface (Ateshian, 1997; Higaki et al., 1997;Schwartz and Hills, 1998).

Articular cartilage is formed during the development of long bonesfollowing the condensation of prechondrocytic mesenchymal cells andinduction of a phenotype switch from predominantly collagen type I tocollagen type II and aggrecan (Hall, 1983; Pechak et al., 1986). Bone isformed from cartilage when chondrocytes hypertrophy and switch to type Xcollagen expression, accompanied by blood vessel invasion, matrixcalcification, the appearance of osteoblasts and bone matrix production.In the adult, a thin layer of articular cartilage remains on the ends ofbones and is sustained by chondrocytes through synthesis, assembly andturnover of extracellular matrix (Kuettner, 1992). Articular cartilagedisease arises when fractures occur due to physical trauma or when amore gradual erosion, as is characteristic of many forms of arthritis,exposes subchondral bone to create symptomatic joint pain (McCarty andKoopman, 1993). In addition to articular cartilage, cartilaginoustissues remain in the adult at several body sites such as the ears andnose, areas that are often subject to reconstructive surgery.

2) Cartilage Repair: The Natural Response

Articular cartilage has a limited response to injury in the adult mainlydue to a lack of vascularisation and the presence of a denseproteoglycan rich extracellular matrix (Newman, 1998; Buckwalter andMankin, 1997; Minas and Nehrer, 1997). The former inhibits theappearance of inflammatory and pluripotential repair cells, while thelatter emprisons resident chondrocytes in a matrix non-conducive tomigration. However, lesions that penetrate the subchondral bone create aconduit to the highly vascular bone allowing for the formation of afibrin clot that traps cells of bone and marrow origin in the lesionleading to a granulation tissue. The deeper portions of the granulationtissue reconstitute the subchondral bone plate while the upper portiontransforms into a fibrocartilagenous repair tissue. This tissue cantemporarily possess the histological appearance of hyaline cartilagealthough not its mechanical properties (Wei et al., 1997) and istherefore unable to withstand the local mechanical environment leadingto the appearance of degeneration before the end of the first yearpost-injury. Thus the natural response to repair in adult articularcartilage is that partial thickness lesions have no repair response(other than cartilage flow and localized chondrocyte cloning) whilefull-thickness lesions with bone penetration display a limited andfailed response. Age, however, is an important factor since fullthickness lesions in immature articular cartilage heal better than inthe adult (DePalma et al., 1966; Wei et al., 1997) and superficiallacerations in fetal articular cartilage heal completely in one monthwithout any involvement of vasculature or bone-derived cells (Namba etal., 1998).

3) Current Approaches for Assisted Cartilage Repair

Current clinical treatments for symptomatic cartilage defects involvetechniques aimed at: 1) removing surface irregularities by shaving anddebridement 2) penetration of subchondral bone by drilling, fracturingor abrasion to augment the natural repair response described above (i.e.the family of bone-marrow stimulation techniques) 3) joint realignmentor osteotomy to use remaining cartilage for articulation 4)pharmacological modulation 5) tissue transplantation and 6) celltransplantation (Newman, 1998; Buckwalter and Mankin, 1997). Most ofthese methods have been shown to have some short term benefit inreducing symptoms (months to a few years), while none have been able toconsistently demonstrate successful repair of articular lesions afterthe first few years. The bone marrow-stimulation techniques of shaving,debridement, drilling, fracturing and abrasion athroplasty permittemporary relief from symptoms but produce a sub-functionalfibrocartilagenous tissue that is eventually degraded. Pharmacologicalmodulation supplying growth factors to defect sites can augment naturalrepair but to date insufficiently so (Hunziker and Rosenberg, 1996;Sellers et al., 1997). Allograft and autograft osteochondral tissuetransplants containing viable chondrocytes can effect a more successfulrepair but suffer from severe donor limitations (Mahomed et al., 1992;Outerbridge et al., 1995).

4) Bone-Marrow Stimulation

The family of bone marrow-stimulation techniques include debridement,shaving, drilling, microfracturing and abrasion arthroplasty. They arecurrently used extensively in orthopaedic clinical practice for thetreatment of focal lesions of articular cartilage that arefull-thickness, i.e. reaching the subchondral bone, and are limited insize, typically less than 3 cm² in area. Use of these procedures wasinitiated by Pridie and others (Pridie, 1959; Insall, 1967; DePalma etal., 1966) who reasoned that a blood clot could be formed in the regionof an articular cartilage lesion by violating the cartilage/boneinterface to induce bleeding from the bone into the cartilage defectthat is avascular. This hematoma could then initiate the classicalcascade of wound healing events that leads to successful healing or atleast scarring in wounds of vascularized tissues (Clark, 1996).Variations of the Pridie drilling technique were proposed laterincluding abrasion arthroplasty (Childers and Ellwood, 1979; Johnson,1991) and microfracturing (Rodrigo et al., 1993; Steadman et al., 1997).Abrasion arthroplasty uses motorised instruments to grind awayabnormally dense subchondral bone to reach a blood supply in the softerdeeper bone. The microfracture technique uses a pick, or an awl, topierce the subchondral bone plate deep enough (typically 3–4 mm), againto reach a vascular supply and create a blood clot inside the cartilagelesion. Practitioners of the microfracture technique claim to observe ahigher success rate than drilling due to the lack of any heat-inducednecrosis and less biomechanical destabilisation of the subchondral boneplate with numerous smaller fracture holes rather than large gaps in theplate producing by drilling (Steadman et al., 1998). Yet another relatedtechnique for treating focal lesions of articular cartilage ismosaicplasty or osteochondral autograft transplantation (OATS) wherecartilage/bone cylinders are transferred from a peripheral “unused”region of a joint to the highly loaded region containing the cartilagelesion (Hangody et al., 1997).

There is no universal consensus among orthopaedists on which type ofarticular cartilage lesion should receive which type of treatment. Thereis also a lack of rigorous scientific studies that demonstrate theefficacy of these treatments for particular indications. Thus the choiceof treatment for cartilage lesions is largely dependent on the training,inclinations and personal experience of the practitioner. Reasons forthis lack of consensus are multifold but include the variability in thetype of lesion treated and a variable if not uncontrolled success in theformation of a “good quality” blood clot. Some of the problemsassociated with forming a good quality blood clot with these proceduresare 1) the uncontrolled nature of the bleeding coming from the bone,which never fills up the cartilage lesion entirely 2) platelet mediatedclot contraction occurring within minutes of clot formation reduces clotsize and could detach it from surrounding cartilage (Cohen et al., 1975)3) dilution of the bone blood with synovial fluid or circulatingarthroscopy fluid and 4) the fibrinolytic or clot dissolving activity ofsynovial fluid (Mankin, 1974). Some of these issues were the motivationbehind some studies where a blood clot was formed ex vivo and then cutto size and packed into a meniscal defect (Arnoczky et al., 1988) or anosteochondral defect (Palette et al., 1992). Something similar to theclassical wound healing cascade then ensued to aid healing of thedefect. This approach did clearly provide more filling of the defectwith repair tissue, however the quality of the repair tissue wasgenerally not acceptable, being predominantly fibrous and mechanicallyinsufficient. Some probable reasons for a less than satisfactory repairtissue with this approach are 1) continued platelet mediated clotcontraction 2) the lack of viability of some blood components due toextensive ex vivo manipulation and 3) the solidification of the clot exvivo which precludes good adhesion to all tissue surfaces surroundingthe cartilage defect and limits defect filling. In summary, currentclinical procedures practised by orthopaedists for treating focallesions of articular cartilage mostly depend on the formation of a bloodclot within the lesion. However the ability to form a good quality bloodclot that fills the lesion and contains all of the appropriate elementsfor wound healing (platelets, monocytes, fibrin network etc) in a viablestate produces inconsistent and often unsatisfactory outcomes. One ofthe embodiments of the present invention ameliorates this situation byproviding a composition and method for delivering these blood bornewound healing elements in a full-volume non-contracting matrix to anarticular cartilage lesion.

5) Biomaterials and Growth Factors

Several experimental techniques have been proposed to repair cartilagelesions using biomaterials and growth factors, sometimes each alone butoften in combination. The analogy with the above-described family ofbone-marrow stimulation techniques is clear. The fibrin scaffold of theblood clot could be replaced with a prefabricated biomaterial scaffoldand the natural mitogenic and chemotactic factors in the blood clotcould be replaced with user-controlled quantities and species of solubleelements such as recombinant growth factors. Examples of this approachinclude the use of fibrin glues to deliver recombinant proteins such asinsulin-like-growth factors (Nixon et al., 1999) and transforming growthfactors (Hunziker and Rosenberg, 1996). Other biologics have beencombined with generic biomaterials such as polylactic acid (PLA),polyglycolic acid (PGA), collagen matrices and fibrin glues includingbone morphogenetic proteins (Sellers et al., 1997; Sellers, 2000; Zhanget al. Patent WO 00/44413, 2000), angiotensin-like peptides (Rodgers andDizerega, Patent WO 00/02905, 2000), and extracts of bone containing amultiplicity of proteins called bone proteins or BP (Atkinson, Patent WO00/48550, 2000). In the latter method, BP soaked collagen sponges neededto be held in the cartilage defect using an additional fibrin/thrombinbased adhesive, creating a rather complex and difficult to reproducewound healing environment. Coating the biomaterial with fibronectin orRGD peptides to aid cell adhesion and cell migration has been done(Breckke and Coutts, U.S. Pat. No. 6,005,161, 1999). Some previousmethods have combined bone-marrow stimulation with post-surgicalinjection of growth hormone in the synovial space with limited success(Dunn and Dunn, U.S. Pat. No. 5,368,051, 1994). Specific biomaterialscompositions have also been proposed such as mixtures of collagen,chitosan and glycoaminoglycans (Collombel et al., U.S. Pat. No.5,166,187, 1992; Suh et al., Patent WO 99/47286, 1999), a crushedcartilage and bone paste (Stone, U.S. Pat. No. 6,110,209, 2000), amulticomponent collagen-based construct (Pahcence et al., U.S. Pat. No.6,080,194, 2000) and a curable chemically reactive methacrylate-basedresin (Braden et al., U.S. Pat. No. 5,468,787, 1995). None of theseapproaches has reached the clinic due to their inability to overcomesome of the following problems 1) lack of retention and adherence of thebiomaterial in the cartilage defect 2) lack of sustained release ofactive forms of these molecules at effective concentrations overprolonged periods of time 3) multiple and uncontrolled biologicalactivities of the delivered molecules 4) cytotoxicity of acidicdegradation products of PGA and PLA 5) inappropriate degradationkinetics or immunogencity of the carrier biomaterial and 6) undesirablesystemic or ectopic affects (calcification of organs) of the activebiologics. The successful implementation of these approaches awaits thesolution to some or all of these issues.

6) Cell Transplantation

Techniques involving cell transplantation have provoked much recentinterest due to their ability to enhance cartilage repair by introducinginto articular defects, after ex vivo passaging and manipulation, largenumbers of autologous chondrocytes (Grande et al., 1989; Brittberg etal., 1994 and 1996; Breinan et al., 1997), allogenic chondrocytes(Chesterman and Smith, 1968; Bently and Greer, 1971; Green, 1977; Astonand Bently, 1986; Itay et al., 1987; Wakatini et al., 1989; Robinson etal., 1990; Freed et al., 1994; Noguchi et al., 1994; Hendrickson et al.,1994; Kandel et al., 1995; Sams and Nixon, 1995; Specchia et al., 1996;Frankel et al., 1997; Hyc et al. 1997; Kawamura et al., 1998), xenogenicchondrocytes (Homminga et al., 1991), perichondrial cells (Chu et al.,1995; Chu et al., 1997), or autogenic and allogenic bone marrow-derivedmesenchymal stem cells (Wakatini et al., 1994; Butnariu-Ephrat, 1996;Caplan et al., 1997; Nevo et al., 1998). The cell transplantationapproach possesses some potential advantages over other cartilage repairtechniques in that they 1) minimise additional cartilage and boneinjury, 2) reduce reliance on donors by ex vivo cell production, 3)could mimic natural biological processes of cartilage development, and4) may provide tailored cell types to execute better repair. Onetechnique using autologous chondrocytes is in the public domain and iscommercially available having been used in several thousand US andSwedish patients www.genzyme.com). In this technique chondrocytes areisolated from a cartilage biopsy of a non-load bearing area,proliferated during several weeks, and re-introduced into the cartilagelesion by injection under a sutured and fibrin-sealed periosteal patchharvested from the patient's tibia. Knowledge of its efficacy has beenquestioned (Messner and Gillquist, 1996; Brittberg, 1997; Newman, 1998)and is unfortunately not known due to the lack of completion of an FDArequested controlled and randomised clinical trial. Recent animalstudies indicate that the injected passaged autologous chondrocytescontribute very little to the observed healing and that the outcome issimilar to that obtained using bone-marrow stimulation (Breinan et al.,1997 and Breinan et al., 2000). Thus the surgical preparation of thedefect could be the main factor inducing repair, in this procedure aswell. Nonetheless, due to the enormous potential benefit of celltransplantation, a large number of patents have been granted in the pasttwo years to protect aspects of autologous chondrocyte processing (Tuboet al., U.S. Pat. No. 5,723,331, 1998; Villeneuve, U.S. Pat. No.5,866,415, 1999), as well as the use and preparation of adipocytes(Mueller and Thaler, U.S. Pat. No. 5,837,235, 1998; Halvorsen et al.,Patent EP 1 077 253, 2001), hematopoeitic precursors (Peterson andNousek-Goebl, U.S. Pat. No. 6,200,606, 2001), amniotic membrane cells(Sackier, 1997), mesenchymal stem cells (Caplan and Hayneworth, U.S.Pat. No. 5,811,094, 1998; Naughton and Naughton, U.S. Pat. No.5,785,964, 1998; Naughton and Willoughby, U.S. Pat. No. 5,842,477, 1998;Grande and Lucas, U.S. Pat. No. 5,906,934, 1999; Johnstone and Yoo, U.S.Pat. No. 5,908,784, 1999), and general techniques usingchondrocytes/fibroblasts and their progenitors, epithelial cells,adipocytes, placental cells and umbilical cord blood cells (Purchio etal., U.S. Pat. No. 5,902,741, 1999), all for use in cartilage repair.

7) The Cell Delivery Problem

Cell transplantation for assisted cartilage repair necessarily involvesa technique to deliver and retain viable and functional transplantedcells at the site of injury. When cells are grown ex vivo with orwithout a support matrix, press-fitting may be used by preparing animplant that is slightly larger than the defect and forcing it therein(Aston and Bentley 1986; Wakatini et al., 1989; Freed et al., 1994; Chuet al., 1997; Frankel et al., 1997; Kawamura et al., 1998).Press-fitting necessitates the use of a tissue that is formed ex vivoand thus not optimised for the geometric, physical, and biologicalfactors of the site in which it is implanted. Suturing or tacking theimplant can aid retention (Sams and Nixon, 1995) although sutures areknown to be an additional injury to the articular surface inducing yetanother limited repair process (Breinan et al., 1997). Biological glueshave been attempted with limited success (Kandel et al., 1995; Jurgensonet al., 1997). When the implant is not amenable to press fitting, suchas with contracting collagen gels or fibrin clots, or when cells alonewithout a support matrix are implanted, often a sutured patch ofperiosteum or another similar tissue is used to retain the implantmaterial within the defect site (Grande et al., 1989; Brittberg et al.,1994; Grande et al., 1989; Brittberg et al., 1996; Breinan et al.,1997). Such a technique may benefit from an ability of the periosteum tostimulate cartilage formation (O'Driscoll et al., 1988 and 1994), butsuffers again from the introduction of sutures and the complex nature ofthe operation involving periosteal harvesting and arthrotomy. Cells havealso been delivered to deep full thickness defects using a viscoushyaluronic acid solution (Robinson et al., 1990; Butnariu-Ephrat, 1996).As with cell sources for cartilage repair, there are several recentlypublished patents for delivery vehicles in cartilage repair ranging fromgel matrices (Griffith et al., 1998; Caplan et al., 1999), to suturesand fibres (Vacanti et al., 1998; Vacanti and Langer, 1998a and 1998b),to screw type devices (Schwartz, 1998), and magnetic systems (Halpern,1997). Taking together the above, current cell delivery techniques forcartilage repair are clearly not optimal. A desirable cell deliveryvehicle would be a polymeric solution loaded with cells which solidifieswhen injected into the defect site, adheres and fills the defect, andprovides a temporary biodegradable scaffold to permit proper celldifferentiation and the synthesis and assembly of a dense, mechanicallyfunctional articular cartilage extracellular matrix.

8) Repair of Other Tissues Including Meniscus, Ligament, Tendon, Bone,Skin, Cornea, Periodontal Tissues, Abscesses, Resected Tumours, andUlcers

Natural and assisted repair of musculoskeletal and other tissues arevery broad fields with numerous complex biological processes and a widevariety of approaches to accelerate the repair process (as in bonerepair), aid it in tissues that have little intrinsic repair capacity(as in cartilage repair), and to reduce scarring (as in burn treatments)(Clark, 1996). Although differences certainly occur in the biologicalelements and processes involved, the global events in (non-fetal) woundrepair are identical. These include the formation of a blood clot at thesite of tissue disruption, release of chemotactic and mitogenic factorsfrom platelets, influx of inflammatory cells and pluripotential repaircells, vascularisation, and finally the resolution of the repair processby differentiation of repair cells their synthesis of extracellularmatrix components. In a successful repair outcome the specific localtissue environment and the specific local population of pluripotentialrepair cells will lead to the formation of the correct type of tissue,bone to replace bone, skin to replace skin etc. Given the similarity ofthe general elements in the tissue repair process, it is not surprisingthat approaches to aid repair in one tissue could also have some successin aiding repair in other tissues. This possibility becomes much morelikely if the method and composition to aid repair is based uponaugmenting some aspect of the natural wound healing cascade withoutsignificantly deviating from this more or less optimised sequence ofevents. In the present invention particular composition and methods areproposed to provide a more effective, adhesive, and non-contractingblood clot at the site of tissue repair. Examples and preferredembodiments are shown for cartilage repair, one of the most difficulttissues to repair. However application of the composition and method andmodifications thereof, conserving the same basic principles, to aidrepair of other tissues including meniscus, ligament, tendon, bone,skin, cornea, periodontal tissues, abscesses, resected tumours, andulcers, are obvious to those who are skilled in the art.

9) Use of Chitosan in Pharmaceuticals, Wound Healing, Tissue Repair andas a Hemostatic Agent

Chitosan, which primarily results from the alkaline deacetylation ofchitin, a natural component of shrimp and crab shells, is a family oflinear polysaccharides that contains 1–4 linked glucosamine(predominantly) and N-acetyl-glucosamine monomers (Austin et al., 1981).Chitosan and its amino-substituted derivatives are pH-dependent,bioerodible and biocompatible cationic polymers that have been used inthe biomedical industry for wound healing and bone induction (Denuziereet al., 1998; Muzzarelli et al., 1993 and 1994), drug and gene delivery(Carreno-Gomez and Duncan, 1997; Schipper et al., 1997; Lee et al.,1998; Bernkop-Schnurch and Pasta, 1998) and in scaffolds for cell growthand cell encapsulation (Yagi et al, 1997, Eser Elcin et al., 1998;Dillon et al., 1998; Koyano et al., 1998; Sechriest et al., 2000; Lahijiet al 2000; Suh et al., 2000). Chitosan is termed a mucoadhesive polymer(Bernkop-Schnurch and Krajicek, 1998) since it adheres to the mucuslayer of the gastrointestinal epithelia via ionic and hydrophobicinteractions, thereby facilitating peroral drug delivery.Biodegradability of chitosan occurs via its susceptibility to enzymaticcleavage by chitinases (Fukamizo and Brzezinski, 1997), lysozymes(Sashiwa et al., 1990), cellulases (Yalpani and Pantaleone, 1994),proteases (Terbojevich et al., 1996), and lipabes (Muzzarelli et al.,1995). Recently, chondrocytes have been shown to be capable ofexpressing chitotriosidase (vasios et al., 1999), the human analogue ofchitosanase; its physiological role may be in the degradation ofhyaluronan, a linear polysaccharide possessing some similarity withchitosan since it is composed of disaccharides of N-acetyl-glucosamineand glucuronic acid.

Chitosan has been proposed in various formulations, alone and with othercomponents, to stimulate repair of dermal, corneal and hard tissues in anumber of reports (Sall et al., 1987; Bartone and Adickes, 1988; Okamotoet al., 1995; Inui et al., 1995; Shigemasa and Minami, 1996; Ueno etal., 1999; Cho et al., 1999; Stone et al., 2000; Lee et al., 2000) andinventions (Sparkes and Murray, U.S. Pat. No. 4,572,906, 1986; Mosbey,U.S. Pat. No. 4,956,350, 1990; Hansson et al., U.S. Pat. No. 5,894,070,1999; Gouda and Larm, U.S. Pat. No. 5,902,798, 1999; Drohan et al., U.S.Pat. No. 6,124,273, 2000; Jorgensen WO 98/22114, 1998). The propertiesof chitosan that are most commonly cited as beneficial for the woundrepair process are its biodegradability, adhesiveness, prevention ofdehydration and as a barrier to bacterial invasion. Other propertiesthat have also been claimed are its cell activating and chemotractantnature (Peluso et al., 1994; Shigemasa and Minami, 1996; Inui et al.,1995) its hemostatic activity (Malette et al., 1983; Malette andQuigley, U.S. Pat. No. 4,532,134, 1985) and an apparent ability to limitfibroplasia and scarring by promoting a looser type of granulationtissue (Bartone and Adickes, 1988; Stone et al., 2000). Although ageneral consensus about the beneficial effects of chitosan in woundhealing is apparent, its exact mechanism of action is not known, nor isthe most effective means of its application, i.e. as a powder,suspension, sponge, membrane, solid gel etc. Part of the reason for theambiguity in its mechanism of action could be that many previous studiesused chitosan that was not chemically defined (acetyl content anddistribution, molecular weight) and of unknown purity. The interestinghemostatic potential of chitosan has also led to its direct applicationto reduce bleeding at grafts and wound sites (Malette et al., 1983;Malette and Quigley, U.S. Pat. No. 4,532,134, 1985). Some studies claimthat the hemostatic activity of chitosan derives solely from it'sability to agglutinate red blood cells (Rao and Sharma, 1997) whileothers believe its polycationic amine character can activate plateletsto release thrombin and initiate the classical coagulation cascade thusleading to its use as a hemostatic in combination with fibrinogen andpurified autologous platelets (Cochrum et al. U.S. Pat. No. 5,773,033,1998). In the context of the present invention, it is important to notein these reports and inventions a complete lack of any example whereblood was mixed with chitosan in solution and applied therapeutically toaid tissue repair through the formation of a chitosan containing blotclot at the repair site.

One technical difficulty that chitosan often presents is a lowsolubility at physiological pH and ionic strength, thereby limiting itsuse in a solution state. Thus typically, dissolution of chitosan isachieved via the protonation of amine groups in acidic aqueous solutionshaving a pH ranging from 3.0 to 5.6. Such chitosan solutions remainsoluble up to a pH near 6.2 where neutralisation of the amine groupsreduces interchain electrostatic repulsion and allows attractive forcesof hydrogen bonding, hydrophobic and van der Waals interactions to causepolymer precipitation at a pH near 6.3 to 6.4. A prior invention(Chenite Patent WO 99/07416; Chenite et al., 2000) has taught thatadmixing a polyol-phosphate dibasic salt (i.e. glycerol-phosphate) to anaqueous solution of chitosan can increase the pH of the solution whileavoiding precipitation. In the presence of these particular salts,chitosan solutions of substantial concentration (0.5–3%) and highmolecular weight (>several hundred kDa) remain liquid, at low or roomtemperature, for a long period of time with a pH in a physiologicallyacceptable neutral region between 6.8 and 7.2. This aspect facilitatesthe mixing of chitosan with cells in a manner that maintains theirviability. An additional important property is that suchchitosan/polyol-phosphate (C/PP) aqueous solutions solidify or gel whenheated to an appropriate temperature that allows the mixed chitosan/cellsolutions to be injected into body sites where, for example cartilagenodules can be formed in subcutaneous spaces in nude mice (Chenite etal., 2000). It is important to note that some other studies haveretained chitosan in a soluble state at physiological pH but thesestudies necessitated the reduction of either chitosan concentration (to0.1% in Lu et al Biomaterials 1999) or of chitosan, molecular weight anddegree of deacetylation (to ˜350 kD and 50% in respectively in Cho et alBiomaterials, 1999) Other studies have also shown that chitosan presentsa microenvironment that supports the chondrocyte and osteoblastphenotype (Suh et al., 2000; Lahiji et al., 2000; Seichrist et al.,2000) however these studies were not based on liquid chitosan in a formthat could be mixed with cells and injected. Finally NN-dicarboxylmethylchitosan sponges have been soaked with BMP7 and placed intoosteochondral defects of rabbits (Mattioli-Belmonte, 1999). Here againsome improved histochemical and immunohistochemical outcome wasobserved, however, incomplete filling of the defect with repair tissueand a significant difficulty in retaining the construct within thedefect appeared to be insurmountable problems. The present inventionovercomes these issues and presents several novel solutions for thedelivery of compositions for the repair of cartilage and other tissues.

10) Summary of Prior Art

In summary of prior art for assisted cartilage repair, it may be saidthat many techniques to improve the very limited natural repair responseof articular cartilage have been proposed and experimentally tested.Some of these techniques have achieved a certain level of acceptance inclinical practice but this has mainly been so due to the absence of anypractical and clearly effective method of improving the repair responsecompared to that found when the family of bone marrow stimulationtechniques is applied. This invention addresses and solves several ofthe main problematic issues in the use of cells and blood components torepair articular cartilage. One main obstacle towards the development ofan effective cartilage repair procedure is the absence of a compositionand method to provide an appropriate macromolecular environment withinthe space requiring cartilage growth (cartilage defect or other siterequiring tissue bulking or reconstruction) This macromolecularenvironment or matrix should 1) be amenable to loading with activebiological elements (cells, proteins, genes, blood, blood components) ina liquid state 2) then be injectable into the defect site to fill theentire defect or region requiring cartilage growth 3) present aprimarily nonproteinaceous environment to limit cell adhesion andcell-mediated contraction of the matrix, both of which induce afibrocytic cellular phenotype (fibrous tissue producing) rather thanchondrocytic cellular phenotype (cartilaginous tissue producing) andwhich can also disengage the matrix from the walls of the defect 4) becytocompatible, possessing physiological levels of pH and osmoticpressure and an absence of any cytotoxic elements 5) be degradable butpresent for a sufficiently long time to allow included biologicallyactive elements to fully reconstitute a cartilaginous tissue capable ofsupporting mechanical load without degradation. In addition it isobvious to those skilled in the art that such a combination ofcharacteristics could be applied with minimal modifications towards therepair of other tissues such as meniscus, ligament, tendon, bone, skin,cornea, periodontal tissues, abscesses, resected tumors, and ulcers.

It would be highly desirable to be provided with a new composition foruse in repair and regeneration of cartilaginous tissues.

SUMMARY OF THE INVENTION

One aim of the present invention is to provide a new composition for usein repair and regeneration of cartilaginous tissues.

In accordance with the present invention, there is thus provided acomposition for use in repair, regeneration, reconstruction or bulkingof tissues of cartilaginous tissues or other tissues such as meniscus,ligament, tendon, bone, skin, cornea, periodontal tissues, abscesses,resected tumors, and ulcers.

In accordance with the present invention, there is also provided the useof a polymer solution that can be mixed with biological elements andplaced or injected into a body site where the mixture aids the repair,regeneration, reconstruction or bulking of tissues. Repaired tissuesinclude for example without limitation cartilage, meniscus, ligament,tendon, bone, skin, cornea, periodontal tissues, abscesses, resectedtumors, and ulcers.

The biological elements are preferably based on blood, blood componentsor isolated cells, both of autologous or non-autologous origin.

Also in accordance with the present invention, there is provided amethod for repairing a tissue of a patient, said method comprising thestep of introducing into said tissue a temperature-dependent polymer gelcomposition such that said composition adhere to the tissue and promotesupport for cell proliferation for repairing the tissue.

The composition preferably comprises at least one blood component.

Still in accordance with the present invention, there is provided amethod for repairing a tissue of a patient, said method comprising thestep of introducing a polymer composition in said tissue, said polymercomposition being mixable with at least one blood component, saidpolymer composition when mixed with said blood component results in amixture, said mixture turning into a non-liquid state in time or uponheating, said mixture being retained at the site of introduction andadhering thereto for repairing the tissue.

The polymer can be a modified or natural polysaccharide, such aschitosan, chitin, hyaluronan, glycosaminoglycan, chondroitin sulfate,keratan sulfate, dermatan sulfate, heparin, or heparin sulfate.

The polymer composition may comprise a natural, recombinant or syntheticprotein such as soluble collagen or soluble gelatin or a polyaminoacids, such as for example a polylysine.

The polymer composition may comprise polylactic acid, polyglycolic acid,a synthetic homo and block copolymers containing carboxylic, amino,sulfonic, phosphonic, phosphenic functionalities with or withoutadditional functionalities such as for example without limitationhydroxyl, thiol, alkoxy, aryloxy, acyloxy, and aroyloxy.

The polymer composition is preferably initially dissolved or suspendedin a buffer containing inorganic salts such as sodium chloride,potassium calcium, magnesium phosphate, sulfate, and carboxylate.

The polymer composition may be dissolved or suspended in a buffercontaining an organic salt such as glycerol-phosphate, fructosephosphate, glucose phosphate, L-Serine phosphate, adenosine phosphate,glucosamine, galactosamine, HEPES, PIPES, and MES.

The polymer composition has preferably a pH between 6.5 and 7.8 and anosmolarity adjusted to a physiological value between 250 mOsm/L and 600mOsm/L.

The blood component may be for example without limitation whole blood,processed blood, venous blood, arterial blood, blood from bone, bloodfrom bone-marrow, bone marrow, umbilical cord blood, or placenta blood.It may also comprise erythrocytes, leukocytes, monocytes, platelets,fibrinogen, thrombin or platelet rich plasma free of erythrocytes.

The blood component can also comprise an anticoagulant such as citrate,heparin or EDTA. To the opposite the blood component can comprise apro-coagulant such as thrombin, calcium, collagen, ellagic acid,epinephrine, adenosine diphosphate, tissue factor, a phospholipid, and acoagulation factor like factor VII to improve coagulation/solidificationat the site of introduction.

The blood component may be autologous or non-autologous.

The polymer composition is preferably used in a ratio varying from 1:100to 100:1 with respect to the blood component.

The polymer composition and the blood component are preferablymechanically mixed using sound waves, stirring, vortexing, or multiplepasses in syringes.

The tissue that can be repaired or regenerated is for example withoutlimitation cartilage, meniscus, ligament, tendon, bone, skin, cornea,periodontal tissue, abscesses, resected tumors, or ulcers. In somecases, the site of introduction in the body may be surgically preparedto remove abnormal tissues. Such procedure can be done by piercing,abrading or drilling into adjacent tissue regions or vascularizedregions to create channels for the polymer composition to migrate intothe site requiring repair.

Further in accordance with the present invention, there is provided achitosan solution for use in cell delivery to repair or regenerate atissue in vivo, said chitosan solution comprising 0.5–3% w/v of chitosanand being formulated to be thermogelling, said solution being mixed withcells prior to being injected into a tissue to be repaired orregenerated. The solution may be induced to thermogel by addition ofphosphate, glycerol phosphate or glucosamine, just to name a few forexample. Preferable, the chitosan solution has a pH between 6.5 to 7.8.

The cells may be selected for example from the group consisting ofprimary cells, passaged cells, selected cells, platelets, stromal cells,stem cells, and genetically modified cells. Preferably the cells aresuspended in a carrier solution, such as a solution containinghyaluronic acid, hydroxyethylcellulose, collagen, alginate, or awater-soluble polymer.

In accordance with the present invention, there is also provided agelling chitosan solution for use in culturing cells in vitro, saidchitosan solution comprising 0.5–3% w/v of chitosan and being formulatedto be thermogelling, said solution being is mixed with cells prior tobeing cultured in vitro.

Preferably, the polymer composition contains between 0.01 and 10% w/v of20% to 100% deacetylated chitosan with average molecular weight rangingfrom 1 kDa to 10 Mda and a blood component.

In accordance with the present invention, there is further provided apolymer composition for use in repairing a tissue, and the use thereof.The composition may also be used for the manufacture of a remedy fortissue repair.

For the purpose of the present invention the following terms are definedbelow.

The terms “polymer” or “polymer solution”, both interchangeable in thepresent application are intended to mean without limitation a polymersolution, a polymer suspension, a polymer particulate or powder, and apolymer micellar suspension.

The term “repair” when applied to cartilage and other tissues isintended to mean without limitation repair, regeneration,reconstruction, reconstitution or bulking of tissues.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1F are schematic representation of the mixing of polymersolution with cells and in vitro solidification and culture forcartilage growth;

FIGS. 2A to 2C illustrate the viability of chondrocytes afterencapsulation and culture in a chitosan/glycerol-phosphate gel;

FIGS. 3A to 3E illustrates cartilage formation within chitosan gel invitro, as measured by glycosaminoglycan (GAG) accumulation;

FIG. 4 illustrates a RNase protection analysis of cartilage-specificmRNAs expressed by primary chondrocytes cultured in chitosan gel for 0,14 and 20 days;

FIG. 5 illustrates a western blot analysis of cartilage-specificproteins expressed by primary chondrocytes cultured in chitosan gel for0, 14 and 20 days;

FIG. 6 illustrates a mechanical behavior of gel discs cultured with andwithout chondrocytes;

FIG. 7 is a schematic representation of polymer mixing with cells andsubcutaneous injection into mice;

FIGS. 8A and 8B illustrate a toluidine blue histology of cartilage grownsubcutaneously in nude mice;

FIG. 9 illustrates a RNase protection analysis of cartilage-specificmRNAs expressed in in vivo implants of chitosan gel with or withoutprimary chondrocytes;

FIG. 10 illustrates a western blot analysis of cartilage-specificproteins expressed in vivo in mouse implants of chitosan gel harboringprimary chondrocytes;

FIG. 11 illustrates the mechanical properties of cartilage implantsgrown subcutaneously in nude mice;

FIGS. 12A and 12B illustrate adhesion of thermogelling chitosan solutionto chondral only defects in ex vivo porcine femoral condyles of intactjoints;

FIGS. 13A and 13B illustrate loading of thermogelling chitosan solutionto chondral defects in rabbits, and 24 hours residence in vivo;

FIG. 14 illustrates the retention of thermogelling chitosan solution inchondral defects in rabbits, 24 hours after injection;

FIG. 15A is a schematic representation showing the preparation, mixingand in vitro solidification of a blood/polymer mixture;

FIGS. 15B and 15C illustrate the liquid blood/polymer solidification invitro, in an agarose well (FIG. 15B) or tube (FIG. 15C) composed ofglass or plastic;

FIG. 16 illustrates an average solidification time of a blood/chitosanmixture versus blood alone using blood from three different species;

FIG. 17A illustrates a clot contraction of blood, or blood/polymermixtures, as measured by plasma release with time, after deposition in aglass vial;

FIGS. 17B and 17C illustrate the physical appearance of solid blood andblood/polymer mixtures, 28 hours post-contraction, in glass tubes (FIG.17B) or as free-swelling discs cast in agarose wells and incubated inTyrode's buffer (FIG. 17C);

FIG. 18 illustrates an admixture of liquid chitosan, but not otherliquid polysaccharide solutions, reversing heparin-mediatedanti-coagulation;

FIGS. 19A to 19C illustrate an histology of blood/polymer mixture;

FIGS. 20A and 20B illustrates viability of leukocytes and plateletsafter mixing with a chitosan solution;

FIG. 21 illustrates a prolonged release of blood proteins from an invitro-formed blood/polymer mixture versus blood alone;

FIGS. 22A to 22C illustrate the preparation, mixing and injection ofpolymer/blood mixture to improve healing of articular cartilage defects;

FIGS. 22D and 22E are a schematic representation of therapy to healhuman articular cartilage;

FIGS. 23A and 23B illustrate enhanced chemotaxis of repair cellsoriginating from bone marrow and migrating towards the cartilage defect,1 week after delivery of the blood/polymer mixture to a chondral defectwith bone-penetrating holes; and

FIGS. 24A and 24B illustrate the growth of hyaline cartilage in defectstreated with a blood/polymer mixture versus growth of fibrotic tissue inuntreated defects.

DETAILED DESCRIPTION OF THE INVENTION

When combined with blood or blood components the polymer could be in anaqueous solution or in an aqueous suspension, or in a particulate state,the essential characteristics of the polymer preparation being that 1)it is mixable with blood or selected components of blood, 2) that theresulting mixture is injectable or can be placed at or in a body sitethat requires tissue repair, regeneration, reconstruction or bulking and3) that the mixture has a beneficial effect on the repair, regeneration,reconstruction or bulking of tissue at the site of placement.

A preferred embodiment is shown in Example 5 where a solution of thenatural polysaccharide, chitosan, was used at a concentration 1.5% w/vand in 0.135 moles/L disodium glycerol phosphate buffer at pH=6.8. Thissolution was mixed with peripheral rabbit blood at a ratio of 1 partpolymer solution to 3 parts blood. The polymer/blood mixture was theninjected into a surgically prepared articular cartilage defect in therabbit where it solidified within 5 minutes (FIG. 22). Histologicalobservations of the healing process revealed a stimulated repair thatresulted in hyaline cartilage after 6–8 weeks (FIG. 24). Control defectsthat did not receive the polymer/blood mixture were incompletely healedor healed with non-functional fibrous or fibrocartilagenous tissue (FIG.24). This example demonstrates that the use of a polymer/blood mixturecan result in more effective healing and greater functionality ofrepaired tissue than simply inducing bleeding at the wound site. Trivialmodifications of this invention are evident to those skilled in the art.Other polymers and other formulations of polymers or polymer blends maybe substituted for the chitosan solution providing they retain the threecharacteristics cited in the previous paragraph. And clearly, thisapproach may be trivially applied to the repair of tissues other thancartilage such as meniscus, ligament, tendon, bone, skin, cornea,periodontal tissues, abscesses, resected tumors, and ulcers.Applications in tissue bulking and reconstruction are also evident.

We present examples and evidence to teach possible mechanisms of actionof this invention including 1) inhibition of the typicalplatelet-mediated contraction of a blood clot by mixing blood with thepolymer prior to solidification (FIG. 17) 2) the resulting maintainedfull-volume scaffold and therefore better defect filling for tissuerepair (FIG. 18) 3) adherence of the solidified polymer/blood mixture tothe surrounding tissues (FIG. 22A) 4) a slower release of chemotacticand mitogenic protein factors from the polymer/blood mixture than from asimple blood clot (FIG. 21) 5) maintenance of leukocyte and plateletviability in the polymer blood/mixture (FIG. 20) and 6) provision of apolysaccharide environment in the repair site that is more conducive tocartilage formation than is a purely proteinaceous matrix (FIGS. 2–6,8–10, 24). These phenomena are demonstrated to occur in our examples.Their demonstration does not, however, reject the possibility that otherimportant events occur such as those involving the kinetics of cellulardegradation of the polymer, and binding/concentration of endogenousfactors by the chitosan.

A second preferred embodiment of this invention is shown in Examples 1and 2 where a thermogelling chitosan solution was used to deliverprimary chondrocytes to subcutaneous regions in mice or to culturechambers in vitro. In this case the absence of blood componentsnecessitates a gelling capability on the part of the chitosan solutionalone, and this property is endowed via a particular preparation of thechitosan solution using glycerol phosphate and other similar buffers. Inour examples we demonstrate that the polymer solution may be mixed withcells and the polymer/cell solution injected in vivo or in vitrowhereupon it gels, maintaining functionality and viability of the cells(FIGS. 1-11). The cells may be resuspended in a physiological buffer, orother cell carrier suspension such as cellulose in an isotonic buffer,prior to mixing with the chitosan solution. We show data demonstratingthe formation of cartilage tissue in vitro (FIGS. 2–6) and in vivo(FIGS. 8–11) when primary chondrocytes are injected with this polymersolution. Trivial modifications and extensions of this embodiment of theinvention are also evident to those skilled in the art where, forexample, other cell types may be used and concentrations of the chitosanand the buffer may be changed to achieve the same result.

The present invention will be more readily understood by referring tothe following examples, which are given to illustrate the inventionrather than to limit its scope.

EXAMPLE 1 Mixing of Thermogelling Chitosan Solution with PrimaryChondrocytes for In Vitro Growth of Cartilage

Chitosan (0.22 g, 85% deacetylated) as an HCl salt powder was sterilizedby exposure to ultraviolet radiation in a biological laminar flow hoodand then dissolved in 7.5 ml H₂O resulting in a pH near 5.0.D(+)-glucosamine (0.215 g, MW 215.6) was dissolved in 10 ml of 0.1M NaOHand filter sterilized using a 22 μm pore size disk filter. Glycerolphosphate (0.8 g, MW 297 including 4.5 mole water per mole glycerolphosphate) was dissolved in 2.0 ml of H₂O and filter sterilized using a22 μm pore size disk filter. 2.25 ml of the glucosamine solution wasadded drop-by-drop under sterile conditions to the chitosan solutionwith agitation at a temperature of 4° C. Then 1 ml of the glycerolphosphate solution was added under the same conditions. This finalsolution is still a liquid and remains so for an extended period (i.e.days) if the temperature is kept low, i.e. near 4° C. The pH of thissolution is physiological at 6.8 and the osmolarity is alsophysiological, around 376 mOsm/kg-H₂O. It is of critical importance toretain these two parameters within the limits required to maintain cellviability. These limits vary with cell type but are generally 6.6<pH<7.8and 250 mOsm/kg-H₂O<osmolarity<450 mOsm/kg-H₂O. A solution is preparedby dissolving 150 mg hydroxyethyl cellulose (Fluka) and 6 ml DMEM(Dulbecco's modified Eagles Medium), and filter sterilized using a 22 μmpore size disk filter. A cell pellet is resuspended with 2 ml ofhydroxyethyl cellulose-DMEM solution, and admixed into thechitosan-glycerol phosphate solution. As a negative control, thechitosan solution mixed with 2 ml of hydroxyethyl cellulose-DMEMsolution with no cells was generated. When this solution is heated to37° C. it transforms into a solid hydrogel similarly to thethermogelling solution disclosed in a previous invention (Chenite et al.Patent WO 99/07416). Most importantly, this previous invention did notdemonstrate that cell viability was maintained throughout thethermogelllng process in this chitosan solution, and thus did not enablethe use of this chitosan solution for cell delivery, tissue repair andtissue regeneration.

The above solution in the liquid state at 4° C. was mixed withenzymatically isolated primary chondrocytes (Buschmann et al., 1992) andthen poured into a plastic culture dish (FIGS. 1A to 1F). In FIG. 1A, acell pellet is resuspended and admixed (FIG. 1B) into the liquidchitosan gel solution at 4° C. In FIGS. 1C and 1D, the liquid solutionis poured into a tissue culture petri and allowed to solidify at 37° C.for 30 minutes, after which the solid gel with cells is washed withDMEM, and discs cored using a biopsy punch. In FIG. 1E, 1000 μm poremesh grids are placed in 48-well plates. In FIG. 1F, the chitosan geldiscs with cells are placed in culture in individual wells.

A gel harboring cells formed after a 20 minute incubation at 37° C.Using a biopsy punch, 6 mm diameter 1 mm thick discs were cored from thegel and placed in culture for up to 3 weeks. Discs were culturedindividually in 48-well tissue culture plates with sterile nylon 1000 μMmeshes beneath to allow media access to all surfaces. Over 90% of theencapsulated cells were viable immediately after encapsulation, andthroughout the culture period (FIGS. 2A to 2C). Samples were incubatedin calcein AM and ethidium homodimer-1 to reveal live (green) and dead(red) cells. Freshly isolated chondrocytes (FIG. 2A) were encapsulatedin the gel, solidified and tested immediately for viability (FIG. 2B),or after 20 days of culture in the gel (FIG. 2C). FIG. 2C shows cellswith typical chondrocyte morphology from the middle of the gel.

Several distinct cell types exhibited the same high degree of viabilityafter encapsulation and cell culture, including Rat-1, COS, 293T, andde-differentiated bovine articular chondrocytes, confirming that thegelation process maintained cell viability, and could thus be used todeliver cells in vivo by injection. Toluidine blue staining of the gelwith cells after 22 days of culture revealed a metachromatic ring ofstaining surrounding encapsulated primary chondrocytes, indicating thebuild-up of proteoglycan, or GAG, which was beginning to fuse betweenclosely adjacent cells (FIG. 3D). These regions also stained withantibodies raised against aggrecan, type II collagen and link protein.The chitosan gel matrix was also found to bind Toluidine blue (FIG. 3C).This property enabled to observe the lattice structure of the gel, afteremploying an aldehyde fixation. Interestingly, the pericellular ring ofGAG observed around the chondrocytes contained little chitosan matrix,the latter appearing to have been degraded by chondrocyte-producedfactors (FIG. 3D). Primary calf chondrocytes were encapsulated inchitosan gel at 2×10⁷ primary chondrocytes per ml and cultured as 6 mmdiscs for up to 20 days. Primary calf chondrocytes were encapsulated andcultured in 2% agarose and analyzed in parallel. Day 0 and day 20cultures were processed by paraffin sectioning and toluidine bluestaining for agarose gel cultures (FIGS. 3A and 3B) and chitosan gelcultures (FIGS. 3C and 3D). At day=0, nuclei stain dark blue (FIGS. 3Aand 3C) whereas accumulated pericellular GAG stains metachromaticblue-violet (FIGS. 3B and 3D, large arrows). These pericellular regionswere immunopositive for aggrecan, collagen (II) and cartilage linkprotein. At a magnification of 40× in FIG. 3E, quantitative biochemicalanalysis of GAG present at days 0, 14, and 20 of culture using the DMMBassay revealed a similar accumulation of GAG in chitosan gel comparedwith agarose gel.

RNA analysis of type II collagen and aggrecan mRNA expressed by theencapsulated chondrocytes revealed high levels at 14 and 22 days ofculture (FIG. 4, lanes 4 and 5) that were comparable to those levelsobserved in articular chondrocytes in cartilage (FIG. 4, lane 6). Amixture of antisense ³²P-labeled RNA probes complementary to bovine typeII collagen, aggrecan, and GAPDH was hybridized with tRNA (lane 1), ortotal RNA, from bovine kidney (lane 2), from primary chondrocytes(10⁷/ml) cultured in chitosan gel for 0 days (lane 3) 14 days (lane 4)or 20 days (lane 5), or adult bovine articular cartilage (lane 6).Samples were treated with RNase A and T1, then submitted toelectrophoresis and autoradiography. Protected bands showing thepresence of individual transcripts are as indicated. The maintenance ofthe chondrocyte phenotype in the chitosan/glycerol-phosphate gel isshown by the continued expression of aggrecan and type II collagen.

Western analysis of proteins produced by encapsulated cells showed anaccumulation of cartilage matrix link protein between 2 and 3 weeks inculture (FIG. 5). Total proteins were extracted, separated by SDS-PAGE,and immunoblotted with antisera recognizing vimentin, PCNA, theC-propeptide of type II collagen, or cartilage link protein. Samplesanalyzed include chitosan gel with no cells (lane 1), bovine kidney(lane 2), duplicate samples of primary chondrocytes (10⁷/ml) cultured inchitosan gel at day=0 (lanes 3 and 4), day=14 (lanes 5 and 6), or day=20(lanes 7 and 8), 2-week calf articular cartilage (lane 9), or adultbovine cartilage (lane 10). Results show the accumulation ofcartilage-specific proteins CP2 and link at 14 and 20 days, as well asthe persistence of PCNA expression through culture day 20, as a markerfor cell proliferation.

Discs containing primary bovine articular chondrocytes were mechanicallyevaluated at days 4 and 13 of culture using uniaxial unconfinedcompression stress relaxation tests. By comparing to control gels withno cells, a significant, cell-dependent degree of stiffening wasobserved even at day 4 and became much more dramatic at day 13 (FIG. 6).Discs (˜5 mm diameter) from days 4, 13 and 19 of culture weremechanically tested in unconfined compression by applying 5 ramps of 10%the disk thickness (˜1.5 mm) during 10 seconds and holding thatdisplacement during subsequent stress relaxation (the 2nd ramp from10–20% is shown in the graph). The gel discs without cells displayed aweak behavior while cell-laden gels became evidently stiffer with timein culture and more characteristically viscoelastic, like articularcartilage.

By analyzing these data with a composite poroelastic model (Soulhat etal., 1999) a doubling of the non-fibrillar matrix modulus (2.5→5 kPa)was found, a 5× increase in the fibrillar matrix modulus (100→500 kPa)was also found together with a near 100× reduction in hydraulicpermeability (5→0.08×10–12 N-s/m4) due to the presence of primarychondrocytes in these gels during only 13 days of culture in vitro.Taken together, these results demonstrate that the chitosan gel iscytocompatible and cytodegradable, conducive to maintenance of thechondrocyte phenotype, and permits the elaboration of a neo-cartilagematrix with a significant increase in mechanical stiffness in vitro.

EXAMPLE 2 Mixing of Thermogelling Chitosan Solution with PrimaryChondrocytes and Subcutaneous Injection for In Vivo Growth of Cartilage

To demonstrate that this in situ gelling system can be employed inanimals, athymic mice (CD1 nu/nu) were subjected to dorsal, subcutaneousinjections of 100 to 300 μl of chitosan gel described in Example 1,containing 10 million calf articular chondrocytes per ml (FIG. 7). Acell pellet of primary calf chondrocytes was admixed with liquidchitosan gel at 4° C. to achieve a concentration of 1 to 2×10⁷ cells/ml,and injected in liquid form as 100 μl subcutaneous dorsal implants inanesthetized nude mice. In situ gelling was apparent by palpation 5 to10 minutes post-injection.

Control mice were similarly injected with chitosan gel alone. A palpablegel was formed within 10 minutes of injection. Implants were recoveredat 21, 48, and 63 days post-injection. Toluidine blue staining revealedthe gross production of GAG-rich extracellular matrix by the implantscontaining cells (FIG. 8A). No GAG accumulation was seen in implants ofchitosan gel alone (FIG. 1B). Primary calf chondrocytes at 2×10⁷cells/ml liquid chitosan gel were injected in liquid form as 100 μlsubcutaneous dorsal implants in anesthetized nude mice. Control micereceived 100 μl subcutaneous dorsal implants of liquid chitosan gelalone. 48 days after injection, implants were harvested and processedfor paraffin histology and toluidine blue staining. Metachromatic violetstaining reveals the accumulation of GAG in the implant withchondrocytes (FIG. 8A). No GAG accumulation is detected in the implantwith chitosan gel only (FIG. 8B).

Cartilage-specific mRNA expression, collagen type II and aggrecan, wasdetected in the in vivo implants with primary chondrocytes at day 48post-injection (FIG. 9).

No type II collagen or aggrecan expression was detected in implants ofchitosan gel alone (FIG. 9). A mixture of antisense ³²P-labelled RNAprobes complementary to bovine type II collagen, aggrecan, and GAPDHwere hybridized with tRNA (lane 1), or total RNA, from bovine kidney(lane 2), from day=48 in vivo nude mouse implants with chitosan gel only(lane 3) or day=48 in vivo implants of chitosan gel with primarychondrocytes at 2×10⁷ cells/ml (lane 4), or adult bovine articularcartilage (lane 5). Samples were treated with RNase A and T1, thensubmitted to electrophoresis and autoradiography. Protected bandsshowing the presence of individual transcripts are as indicated. Themaintenance in vivo of the chondrocyte phenotype in thechitosan/glycerol-phosphate gel is shown by the expression of aggrecanand type II collagen.

Cartilage-specific proteins were detected in in vivo implants withprimary chondrocytes from days 48 and 63 post-injection (FIG. 10). Nocartilage-specific proteins were detected in implants with chitosan gelonly (FIG. 10). Total proteins were extracted, separated by SDS-PAGE,and immunoblotted with antisera recognizing vimentin, PCNA, theC-propeptide of type II collagen, or cartilage link protein. Samplesanalysed include chitosan gel with no cells (lane 1), bovine kidney(lane 2), two distinct in vivo nude mouse implants of chitosan gel onlyat day 63 (lanes 3 and 4), of in vivo implants of chitosan gel with2×10⁷ calf chondrocytes per ml gel at days 48 (lane 5) or day 63 (lane6), 2-week calf cartilage (lane 7), or adult bovine cartilage (lane 8).Results show the accumulation of cartilage-specific extracellular matrixproteins CP2 and link, in only those chitosan gel implants carryingchondrocytes. The acronym PCNA means “proliferating cell nuclearantigen”. CP refers to type 2 collagen C pro-peptide and link refers tocartilage link protein.

The in vivo implants with no cells had a pasty consistency, whereas theimplants with cells could be cored into 3 to 5 mm discs and subjected tomechanical testing to reveal a high mechanical stiffness not found in anin vitro disc without cells (FIG. 11). These data indicate thatchondrocytes can be delivered in situ, via injection, with the chitosanthermogelling solution as a carrier. The injected chondrocytes remainviable, and synthesize and assemble significant levels of aproteoglycan-rich extracellular matrix that stiffens over time to form afunctional cartilaginous tissue. In FIG. 11, primary calf chondrocytesat 2×10⁷ cells/ml liquid chitosan gel were injected in liquid form as100 μl subcutaneous dorsal implants in anesthetized nude mice. Controlmice received 100 μl subcutaneous dorsal implants of liquid chitosan gelalone. 48 days after injection, implants were harvested. Implants ofchitosan gel only had a paste-like consistency, and could not bemechanically tested. Implants with primary chondrocytes had theappearance of cartilage, and a 3 mm biopsy was cored from the center ofthe implant, and tested in unconfined compression using 2.5% thicknesscompression with a relaxation criteria of 0.05 g/min. The equilibriummodulus at 20% and 50% compression offset is shown for the 48 dayimplant containing cells compared to a control disk left in vitro duringa 42 day period. The in vivo grown chondrocyte laden gel has developedsubstantial mechanical stiffness during 48 days due to the synthesis andassembly of a functional cartilage matrix (FIG. 8A).

EXAMPLE 3 Adhesion of Thermogelling Chitosan Solution to Cartilage andBone Surfaces

One of the most significant advantages of this chitosan thermogellingformulation for cartilage repair is its ability to conform and adhere toirregular cartilage defects and other irregularly shaped cavities in thebody that require tissue repair, regeneration, reconstruction orbulking. Many current tissue repair procedures suffer drastically inthis respect. Chitosan-glycerol phosphate liquid gel without cells wasdelivered ex vivo to porcine femoral condylar intra-chondral (notinvolving bone) defects. Disc-shaped defects in the articular cartilagewere created using a biopsy punch (FIG. 12A) and the chitosan solutiondescribed in Example 1 was injected into these defects and allowed tosolidify in an incubator at 37° C. The articulating cartilage surfacewas opposed and simulated joint motions were performed after which thegel was observed to remain in the cartilage defect (FIG. 12B). The gelnot only remained in the defect but also adhered to the surrounding boneand cartilage surfaces and did not contract. In FIGS. 12A and 12B,liquid chitosan gel was deposited in 6 mm diameter full-thicknesscartilage defects (FIG. 12A) and allowed to solidify at 37° C. for 30minutes in a humidified incubator. The joint was then closed, and jointmotion simulated for several minutes. The chitosan gel adhered to andwas retained in all of the defects after simulated joint motion (FIG.12B).

In vivo filling of intrachondral defects was also performed on thepatellar groove of rabbits. A rectangular (4 mm×5 mm) defect was createdby shaving off cartilage down to the harder calcified cartilage layerwith a microsurgical knife. Several microfracture holes were introducedusing a 16-gauge needle, The thermogelling chitosan solution describedin Example 1 was injected into this defect and allowed to solidify for 5minutes (FIG. 13A) and the rabbit knee joint sutured up. The rabbit wasallowed to ambulate freely and the following day it was euthanised andthe treated knee joint prepared for histological analysis (FIG. 13B). Alive New Zealand White rabbit was anesthetized, and a 3×4 mmchondral-only defect created in the trochlea of the femoral patellargroove. Several microfracture holes were introduced with a 16 gaugeneedle. Liquid thermogelling chitosan was loaded into the defect andallowed to gel for 5 minutes in situ (FIG. 13A). The joint was closed,and the rabbit allowed to recover with unrestricted motion for 24 hoursbefore sacrifice and joint dissection (FIG. 13B).

Histological analysis (FIG. 14) revealed the retention of thisthermogelling chitosan gel in the very thin cartilage layer of therabbit (only about 0.8 mm thick). The gel adhered firmly to surroundingbone and cartilage tissue, demonstrating good retention, therebyenabling its use as an injectable thermogelling polymer delivery vehiclefor the repair of cartilage and other tissues. The joint and defectshown in FIG. 13B (filled with thermogelling chitosan, and residing 24hours in vivo) was fixed, embedded in LR White plastic resin, sectioned,and stained with Toluidine Blue. A cross-section of the defect revealsretention of the chitosan gel in situ, as well as adherence to cartilageand bone surfaces in the defect.

EXAMPLE 4 Preparation, Mixing and In Vitro Solidification ofBlood/Polymer Mixture

Several distinct mixing methods were employed to admix blood with anaqueous polymer solution (FIG. 15A). Blood and polymer are admixed in arecipient, resulting in a homogenous liquid blend of blood and polymer.

In general, 3 volumes blood was mixed with 1 volume of 1.5%polysaccharide in an isotonic and iso-osmolaric solution. In the case ofchitosan gel, 1.5% chitosan was dissolved in 70 mM HCl and 135 mMβ-glycerol phosphate. In the first blood/polymer mixing method, one, 1cc syringe was loaded with 750 μl whole peripheral blood, and a second 1cc syringe was loaded with 250 μl liquid polymer solution. The syringeswere interconnected, and mixed by pumping the two phases back-and-forth40 times, until apparently homogenous. In the second mixing method, 625μl of liquid polymer solution was deposited in a 2.0 ml cryovial(Corning) with several 3 mm–6 mm steel balls. The cryovial was filledwith 1.875 ml whole blood, the cap screwed on, and the vial shakenvigorously for 10 seconds. In the third mixing method, 2 ml of liquidpolymer solution was deposited in a sterile 12 ml glass borosilicatevial (InterGlass 5 cc serological vial). The vial closed with a rubberstopper and metal crimper, and a 25 ml air vacuum was drawn in the vialwith a 10 ml syringe and 20-gauge needle. Using proper phlebotomytechniques, peripheral blood from either rabbit artery, or human orequine vein was drawn into a sterile 10 ml syringe. A 20-gauge needlewas attached to the syringe, and inserted through the rubber stopper ofthe vial. 6 ml of peripheral blood was admitted to the vial. The vialwas vortex mixed for 10 seconds at full speed. Following any of thesemixing techniques, the resulting mixture was deposited into a 4 mlborosilicate glass vial at room temperature, a plastic vial at 37° C.,or an agarose well (FIGS. 15B and 15C), or an articular cartilage defectex vivo. As a control, the same treatment was performed with peripheralwhole blood only. As another control, a vacutainer vial of EDTA-treatedblood was drawn to measure CBC and platelet number. All blood samplestested displayed normal CBC and platelet counts for the respectivespecies. Regardless of the species, the prepared blood/polymer,solidified and adhered strongly to the walls of the glass vial within2.5 to 18 minutes after mixing (FIG. 16). Mixed whole peripheral bloodsolidified in general more slowly compared to blood/chitosan gel (FIG.16). Separate samples of blood, with or without liquid chitosan gel,were mixed and solidification time was measured by the number of minuteselapsed between mixing, and achieving a solid adherent mass in theoriginal mixing vial, or secondary recipient.

Testing of additional blood/polymer solutions, includingblood/hyaluronic acid, blood/hydroxyethyl cellulose, and blood/alginate,revealed that these mixtures also solidify in a time period that iscomparable to blood alone (FIG. 17A). Here it was concluded thatadmixture of chitosan liquid gel into whole peripheral blood acceleratesclot formation, and that blood/chitosan gel solidification time isacceptable for clinical application. Contraction was tested on mixedfresh peripheral rabbit blood, or rabbit blood mixed with PBS or various1.5% polysaccharide solutions including chitosan in glycerol phosphatebuffer. Fresh blood without mixing was also analyzed. A heparinblood/chitosan in glycerol phosphate buffer mixture was also analyzed.500 μl of each sample was deposited into a 4 ml glass tube at 37° C. Atdistinct time points, all excluded plasma was removed from each tube andweighed, to determine the amount of clot contraction. All samples exceptblood/chitosan glycerol phosphate mixtures contracted to 30–50% of theiroriginal volume. Blood/chitosan mixtures contracted minimallymaintaining approximately 90% of their initial volume.

To test for the degree of contraction of solidified blood/polymer mixesrelative to coagulated whole blood, a clot contraction test wasperformed on an array of blood/polymer samples, using several controls(FIGS. 17A, 17B and 17C). One group of controls consisted ofnon-agitated whole peripheral blood, or agitated whole peripheral blood,or whole peripheral blood agitated 3:1 (volume:volume) withphosphate-buffered saline. These samples were compared with experimentalsamples containing 3 volumes whole peripheral blood agitated with 1volume of distinct 1.5% polysaccharide solutions dissolved in PBS(alginate, hydroxyethyl cellulose, or hyaluronic acid). Another sampleconsisted of 3 volumes whole peripheral blood mixed with 1 volumechitosan-glycerol phosphate solution. At intervals up to 18 hours aftersolidification, the excluded serum for each condition was measured intriplicate, as an indication of degree of contraction. Samples withperipheral blood, ±PBS, contracted to 30% of the original mass (FIG.17A). Peripheral blood admixed with the polysaccharides alginate,hydroxyethyl cellulose, or hyaluronic acid contracted to 40%–50% of theoriginal mass (FIG. 17A). The blood/chitosan gel samples showednegligible contraction, with contraction to 90% of the original mass(FIG. 17A). The heparinised blood/chitosan gel samples also resistedcontraction, to 85% of the original mass (FIG. 17A). From these data itwas concluded that blood/chitosan gel resists contraction, and providesa more space-filling fibrin scaffolding inside the cartilage defect. InFIGS. 17B and 17C, samples shown include blood (1), or mixed blood (2),blood/PBS (3), blood/chitosan in glycerol-phosphate (4), heparinblood/chitosan (S), blood/alginate (6), blood/hydroxyethyl cellulose(7), and blood/hyaluronic acid (8).

To test whether anti-coagulated blood could be used to generateblood/polysaccharide in situ solidifying implants, 3 volumes of bloodtreated with 1.5 mM EDTA, 0.38% citrate, acid-0.38% citrate dextrose, orsodium heparin (Becton Dickinson) was mixed with 1 volumechitosan-glycerol phosphate solution. Chitosan-glycerol-phosphatesolution was able to reverse heparin- (FIG. 18), EDTA-, andcitrate-mediated anti-coagulation. 1.5% chitosan in glycerol-phosphatesolution, or three distinct 1.5% polysaccharide solutions, were admixedat a ratio of 1 volume polysaccharide solution, to 3 parts wholeperipheral blood. 500 μl of each sample was deposited in a glassborosilicate tube and allowed to solidify for 60 minutes at 37° C.Different polysaccharides include hyaluronic acid-PBS (1), hydroxyethylcellulose-PBS (2), alginate-PBS (3), and chitosan-glycerol phosphate(4). As a control, heparin blood only was analyzed (5). After 60minutes, the tubes were laid horizontally and photodocumented. Only themixture of chitosan-glycerol phosphate and heparinised blood becamesolid.

Other heparin blood/polysaccharide mixtures using hydroxyethylcellulose, alginate, or hyaluronic acid, failed to solidify (FIG. 18).From these data it was concluded that blood/chitosan in situ solidifyingimplants can be generated using anti-coagulated blood.

Histological sections of solid blood/polymer samples showed thatmixtures were homogenous, that red blood cells did not hemolyse aftermixing or solidification, and that platelets became activated and werefunctional (as evidenced by the generation of a dense fibrin network)(FIGS. 19A to 19C). A solidified mixture of blood/chitosan was fixed,embedded in LR White plastic, sectioned, and stained with ToluidineBlue. (In FIG. 19A, at 20× magnification, global homogeneous mixing isapparent. In FIG. 19B, at 100× magnification, intermixed pools of redblood cells and chitosan hydropolymer is apparent. At 2000×magnification (by environmental electron scanning microscopy) thepresence of fibrin fiber network throughout the blood/chitosan compositeis evident.

Some leukocytes remained viable a number of hours following mixing andsolidification (FIG. 20). Peripheral whole blood was mixed with chitosangel and allowed to solidify. In FIG. 20A, 60 minutespost-solidification, the plug was placed in viability stain with calceinAM/ethidium homodimer-1 to reveal live white blood cells (green cells,large arrows), live platelets (green cells, small arrows), and deadwhite blood cells (red nuclei). In FIG. 20B, a distinct sample was fixedat 180 minutes post-solidification, embedded in LR-White, and submittedto Transmission Electron Microscopy. Active phagocytosis by peripheralmonocytes (arrow head), reflecting cell viability, is evident in TEMmicrographs at 3 hours post-mixing and solidification.

An analysis of the total serum proteins lost from either blood orblood/chitosan following solidification was performed. Equal volumes ofblood, or blood/chitosan gel were solidified in agarose wells. The discswere transferred to individual wells of a 48-well plate containing 1 mlPBS and incubated at 37° C. for 3 hours. The discs were successivelychanged into fresh PBS solution at 37° C. at 4, 5, 7, and 19 hours. PBSwashes were lightly centrifuged to remove any cells prior to analysis.Several discs were extracted for total protein after 3 or 19 hours inPBS. Total proteins present in the discs, or PBS washes, were analysedby SDS-PAGE and total protein stain with Sypro Orange. Serum proteinswere released more slowly more sustained from the blood/chitosan samplescompared with blood samples (FIG. 21). These data suggest that blood andplatelet-derived proteins involved in wound healing are released in amore sustained and prolonged manner from blood/chitosan-filled defects,compared with blood clot-filled defects. Solid discs of blood/chitosangel, or blood only, were generated from 150 μl initial liquid volume.Resulting discs were washed in 1 ml PBS for 3 hours, then transferredsuccessively at 4, 5, 7, and 19 hours for a total of four additional 1ml PBS washes. After 3 or 19 hours of washing, representative discs wereextracted with GuCl to solubilise total retained proteins. Solubleproteins were precipitated from equal volumes of GuCl extracts or PBSwashes, separated on SDS-PAGE gels, and stained for total proteins usingSypro Orange. Comparatively, more proteins were retained in theblood/polymer discs than the blood discs throughout the 19 hour washperiod. Comparatively, a slower and more prolonged release of serumproteins into the PBS washes was seen for blood/chitosan than blood overthe 19 hour wash period.

EXAMPLE 5 Preparation, Mixing and Injection of Blood/Polymer Mixture toImprove Healing of Articular Cartilage Defects

Chondral defects with perforations to the subchondral bone were treatedwith a peripheral blood/chitosan-glycerol phosphate mixture that wasdelivered as a liquid, and allowed to solidify in situ (FIGS. 22A to22C). In FIG. 22A, a full-thickness cartilage defect, 3×4 mm square, wascreated in the femoral patellar groove of an adult (more than 7 months)New Zealand White rabbit. Four, 1 mm diameter microdrill holes werepierced to the bone, until bleeding was observed. In FIG. 22B, liquidwhole blood was mixed at a ratio of 3 volumes blood to 1 volume chitosanin glycerol phosphate solution, and deposited to fill the defect. InFIG. 22C, after 5 minutes in situ, the blood/chitosan implant appearedto solidify. The capsule and skin were sutured, and the animal allowedto recover with unrestricted motion.

A similar treatment in human patients is schematized in FIG. 22D, whereprepared cartilage defects receive an arthroscopic injection of liquidblood/polymer that solidifies in situ. Alternatively, an arthroscopicinjection of liquid polymer is mixed with bone-derived blood at thedefect site (FIG. 22E). In FIG. 22D, the patient blood is mixed with thepolymer ex vivo, and delivered to a prepared defect by arthroscopicinjection, or (FIG. 22E) the polymer is delivered arthroscopically orduring open knee surgery and mixed at the defect site with patient bloodissuing from the defect.

As a proof-of-concept study, the effects of blood/chitosan gel treatmentwere tested in rabbits. Adult, skeletally mature New Zealand Whiterabbits (7 months and older) were anesthetized, with xylazine-ketaminefollowed by isofluorene/oxygen gas anesthesia. The trochlea of thefemoral patellar groove was exposed by a parapatellar incision andpatellar displacement. A full-thickness cartilage defect, up to 4×5 mm,in the trochlea of the femoral patellar groove, was produced with amicrosurgical knife. Four, 4 mm deep, 1 mm diameter bone-penetratingholes were generated by either microdrill with constant irrigation with4° C. PBS, or by puncture with a custom-made awl and hammer. The defectwas flushed with PBS, and depending on the degree of bleeding, up to 200μl of sterile epinephrine (2 μg/ml) in phosphate buffered saline wasinjected into the bleeding holes. The cartilage defect was covered witha sterile gauze soaked with PBS. Rabbit peripheral blood was removedfrom the central artery of the ear with a vacutainer™ needle anduntreated, siliconized glass 4 cc vacutainer™ vials from BectonDickison.

In one treatment, 750 μl blood was drawn into a sterile 1 cc syringe. Asecond syringe holding 250 μl of chitosan-glycerol phosphate solution(1.5% chitosan/70 mM HCl/135 mM β-glycerol phosphate) was interconnectedwith the blood-containing syringe with a sterile plastic connector. Thesyringes were pumped back-and-forth 40 times. The mix was drawn into onesyringe, to which a 20-gauge needle was attached. After purging half ofthe mix, one drop (about 25 μl) was deposited into the defect. In aseparate treatment, 2 ml blood was added to a polypropylene cryovialtube containing 667 μl 1.5% chitosan/70 mM HCl/135 mM β-glycerolphosphate and 6 sterile 3.2 mm diameter stainless steel beads. The tubewas capped, and shaken for 10 seconds, rigorously (around 40 to 50actions). The resulting liquid blood/chitosan mix was removed from thevial with a sterile 1 cc syringe, and a 20 g needle was attached to thesyringe. After purging 200 μl from the syringe, one drop (about 25 μl)was deposited to fill the cartilage defect. The blood/chitosan mixturewas allowed to solidify for 5 minutes, after which the capsule and skinwere sutured, and the wound disinfected. Rabbits were sacrificed at 1week (n=1, male) or at 51 or 56 days (n=2, 1 male, 1 female). Jointswere fixed, decalcified, embedded in LR/White plastic, sectioned, andstained with Toluidine Blue. Blood/chitosan-treated defects at 1 week ofhealing revealed large numbers of chemotactic cells migrating towardsthe blood/chitosan-filled zone (FIG. 23A). Untreated defects had arelatively weak chemotactic response (FIG. 23B) towards the blood clotat the top of the defect. A chondral defect with microdrill holes wascreated in both femoral patellar grooves of an adult New Zealand Whiterabbit, one of which was filled with blood/chitosan gel, and anotherleft untreated. One week after healing, the joints were fixed, processedin LR-White, and Toluidine blue stained. At 2 to 3 mm below the surfaceof the cartilage, a large number of cells migrating towards the defectfilled with blood/chitosan were evident (FIG. 23A), whereas fewermigrating cells were seen at the same region of the untreated defect(FIG. 23B).

After 5 to 8 weeks healing, the blood/chitosan-treated defect was filledwith hyaline repair tissue in 2 rabbits (1 male, 1 female)(FIG. 24A).This blood/chitosan-based repair tissue had the appearance of hyaline,GAG-rich cartilage repair tissue. The repair tissue from untreated, orblood-only treated microfracture defects, had the appearance offibrocartilage (FIG. 24B). There was no histological evidence ofblood/chitosan or blood clot persisting within the defect site at orbeyond 3 weeks post-delivery. A chondral defect with microdrill holeswas created in both femoral patellar grooves of an adult New ZealandWhite rabbit, one of which was filled with blood/chitosan gel, andanother left untreated. At 51 or 56 days after healing, the joints werefixed, processed in LR-White, and Toluidine blue stained. In FIG. 24A,repair tissue from the blood/chitosan-treated defect had the appearanceof metachromatically staining hyaline cartilage, which adhered to thedefect surfaces, and filled the defect. In FIG. 24B, repair tissue fromthe untreated defect had the appearance of fibro-cartilage, withpractically no metachromatic staining for GAG, and only partial defectfilling.

While the invention has been described with particular reference to theillustrated embodiments, it will be understood that numerousmodifications thereto will appear to those skilled in the art.Accordingly, the above description and accompanying drawings should betaken as illustrative of the invention and not in a limiting sense. Forexample, we have demonstrated that mixing chitosan in solution withblood allows the formation of polymer/blood clot that does not contractsignificantly, demonstrates a slowed release of chemotactic andmitogenic blood proteins, maintenance of blood cell viability, and adramatically improved repair of articular cartilage defects. It isobvious to those skilled in the art that the chitosan solution could beprepared differently to achieve the same result. Examples include: 1)altered chitosan concentration and mixing ratio with blood 2) alteredchoice of aqueous solution by changing buffer type and speciesconcentration 3) an aqueous suspension of chitosan aggregates 4) aparticulate chitosan powder combined with a proper mixing technique todistribute these particle throughout the blood and partly dissolve them.Other polymers may be used such as 1) another polysaccharide likehyaluronan if its anti-coagulant effect is overcome by formulating it ina procoagulating state (such as by using a low concentration orcombining it with thrombin) and 2) a protein polymer such as polylysineor collagen could be used to achieve similar effects. Although it is notbelieved that these latter approaches will be as successful as ourpreferred embodiment, due to immunogenicity, toxicity, and celladhesion/contraction effects, these and other formulations areconsidered part of the present invention since they possess thecharacteristics of the polymer preparation of the present inventionbeing that 1) it is mixable with blood or selected components of blood,2) that the resulting mixture is injectable or can be placed at or in abody site that requires tissue repair, regeneration, reconstruction orbulking and 3) that the mixture has a beneficial effect on the repair,regeneration, reconstruction or bulking of tissue at the site ofplacement.

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1. A method for repair and/or regeneration in cartilaginous tissuecomprising administering at a site of the cartilaginous tissue in needof repair an effective amount of a polymer composition comprising: asolution of a polymer; and blood, wherein the polymer includes at leastone selected from the group consisting of a polysaccharide, a protein,and a polyamino acid, and further wherein when the polymer is combinedwith blood the polymer composition is converted into a non-liquid statein time or upon heating such that the polymer compositions when placedat the site in need of repair, the polymer composition will adhere tothe site in need of repair to effect reconstruction or bulking of thetissue and/or regeneration thereof.
 2. The method of claim 1, whereinthe polysaccharide is a modified or natural polysaccharide.
 3. Themethod of claim 2, wherein the polysaccharide is selected from the groupconsisting of chitosan, chitin, hyaluronan, glycosaminoglycan,chondroitin sulfate, keratan sulfate, dermatan sulfate, heparin, andheparin sulfate.
 4. The method of claim 1, wherein the polysaccharide ischitosan.
 5. The method of claim 1, wherein cartilaginous tissue isselected from the group consisting of cartilage, meniscus, ligament andtendon.
 6. The method of claim 1, wherein the protein is a natural,recombinant or synthetic protein.
 7. The method of claim 3, wherein saidprotein is a polyamino acid.
 8. The method of claim 7, wherein thepolyamino acid is a polylysine.
 9. The method of claim 6, wherein thenatural protein is soluble collagen or gelatin.
 10. The method of claim1, wherein the polymer composition is dissolved or suspended in a buffercontaining organic or inorganic salts.
 11. The method of claim 10,wherein the inorganic salts are selected from the group consisting ofsodium chloride or phosphates, sulfates or carboxylates of potassium,calcium and magnesium.
 12. The method of claim 1, wherein said polymercomposition has a pH between 6.5 and 7.8.
 13. The method of claim 1,wherein said polymer solution has an osmolarity adjusted to aphysiological value between 250 mOsm/L and 600 mOsm/L.
 14. The method ofclaim 1, wherein the blood is anticoagulated.
 15. The method of claim 1,wherein the blood further comprises an anticoagulant selected from thegroup consisting of citrate, heparin or EDTA.
 16. The method of claim 1,wherein the blood further comprises a pro-coagulant to improvecoagulation/solidification at the site of introduction.
 17. The methodof claim 1, wherein the pro-coagulant is selected from the groupconsisting of thrombin, calcium, collagen, ellagic acid, epinephrine,adenosine diphosphate, tissue factor, a phospholipid, and a coagulationfactor.
 18. The method of claim 1, wherein the blood is autologous ornon-autologous.
 19. The method of claim 18, wherein the blood is free ofat least one of erythrocytes and/or leukocytes.
 20. The method of claim18, wherein the blood is enriched in platelets.
 21. The method of claim1, wherein the polymer composition is used in a ratio varying from 1:100to 100:1 with respect to the blood.
 22. The method of claim 1, whereinsaid polymer and blood are mechanically mixed using sound waves,stirring, vortexing, or multiple passes in syringes.
 23. The method ofclaim 10, wherein the organic salts are selected from the groupconsisting of glycerol-phosphate, fructose phosphate, glucose phosphate,L-serine phosphate, adenosine phosphate, glucosamine, galactosamine,HEPES, PIPES and MES.
 24. The method of claim 1, wherein the blood isselected from the group consisting of whole blood, processed blood,venous blood, arterial blood, blood from bone-marrow, umbilical cordblood and placenta blood.
 25. The method of claim 17, wherein thecoagulation factor is factor VII.
 26. The method of claim 4, wherein thechitosan is dissolved in an organic or inorganic phosphate buffer. 27.The method of claim 26, wherein the organic or inorganic phosphatebuffer is a phosphate or glycerol phosphate containing buffer.
 28. Themethod of claim 26, wherein the chitosan in the composition is in asoluble state, said composition having a pH between 6.5 and 7.4.
 29. Themethod of claim 1, wherein the cartilaginous tissue is selected from thegroup consisting of articular cartilage, nose cartilage, ear cartilage,meniscus and avascular cartilage.
 30. A method for repair and/orregeneration in cartilaginous tissue comprising administering at a siteof the cartilaginous tissue in need of repair an effective amount of athermogelling composition comprising: a solution of the polymerchitosan; glycerol phosphate; and blood, wherein the thermogellingcomposition is converted into a non-liquid state in time or uponheating, said composition once converted into a non-liquid state adheresto the site in need of repair when placed thereon to effectreconstruction or bulking of the tissue and/or regeneration thereof. 31.The method of claim 30, wherein cartilaginous tissue is selected fromthe group consisting of cartilage, meniscus, ligament and tendon. 32.The method of claim 30, wherein said polymer composition has a pHbetween 6.5 and 7.8, and wherein said chitosan is in solution at saidpH.
 33. The method of claim 30, wherein said polymer solution has anosmolarity adjusted to a physiological value between 250 mOsm/L and 600mOsm/L.
 34. The method of claim 30, wherein the blood is anticoagulated.35. The method of claim 30, wherein the blood further comprises ananticoagulant selected from the group consisting of citrate, heparin orEDTA.
 36. The method of claim 30, wherein the blood further comprises apro-coagulant to improve coagulation/solidification at the site ofintroduction.
 37. The method of claim 30, wherein the pro-coagulant isselected from the group consisting of thrombin, calcium, collagen,ellagic acid, epinephrine, adenosine diphosphate, tissue factor, aphospholipid, and a coagulation factor.
 38. The method of claim 30,wherein the blood is autologous or non-autologous.
 39. The method ofclaim 38, wherein the blood is free of at least one of erythrocytesand/or leukocytes.
 40. The method of claim 38, wherein the blood isenriched in platelets.
 41. The method of claim 30, wherein saidchitosan, glycerol phosphate and blood are mechanically mixed usingsound waves, stirring, vortexing, or multiple passes in syringes. 42.The method of claim 30, wherein the blood is selected from the groupconsisting of whole blood, processed blood, venous blood, arterialblood, blood from bone-marrow, umbilical cord blood and placenta blood.43. The method of claim 37, wherein the coagulation factor is factorVII.
 44. The method of claim 30, wherein the cartilaginous tissue isselected from the group consisting of articular cartilage, nosecartilage, ear cartilage, meniscus and avascular cartilage.